Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33–mega–base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

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Figs. S1 to S8 Legends for data S1 to S5
Other Supplementary Material for this manuscript includes the following:

Figure S2 :
Figure S2: Screening and validation of chr8pLOH cell clones.(A) Schematic illustration of dual-guided chromosome engineering using CRISPR/Cas9 technology.(B) Agarose gel images of the PCR products using primer pairs flanking the targeted region at 2 Mb and 35 Mb of chr8p.PCR product size is expected at 1500 bp or lower depending on the breakpoint repair.(C) Sanger sequencing results of PCR products using primer pairs flanking the targeted region of the deleted allele.Shown are the sequencing chromatograms.In addition, sequencing of the undeleted alleles by primers flanking each cut site (2 Mb and 35 Mb) revealed alterations, as indicated in the box, at each cut side individually.(D) Representative multiplex FISH of chr8pWT and chr8pLOH clones of HLF, HLE and HCC68 cells.Chromosome 8 is colored in light grey and highlighted with a white rectangle (WT) or circle (LOH).(E) WES-based paired copy number analysis of chr8 in HLF, HLE and HCC68 cells relative to their isogenic cell clone pair depicted as rainfall plots.(F) Brightfield microscopy images showing chr8pWT and chr8pLOH cell clone morphology in HLF, HLE and HCC68 cells.(G) Crystal violet stain of HLF, HLE and HCC68 cells before Cas9-Blast transduction and Cas9expressing chr8pWT and chr8pLOH clones after selection with 2 µg/mL Puromycin (Puro) or 10 µg/mL Blasticidin (Blast).

Figure S3 :
Figure S3: Chr8pLOH is associated with metastasis.(A) Scatter plot showing ratios of patients with chr8p-deleted tumors at metastatic and primary sites.Data obtained from the Hartwig Medical Foundation dataset (27).(B) Frequency of patients with chr8pLOH or chr8pWT at primary site, local and distant metastases of selected cancer entities.(C) Proliferation of chr8pLOH cell clones determined by BrdU incorporation ELISA relative to respective chr8pWT clones.Data are represented as mean ± SD of two to three independent experiments.Single dots represent technical replicates.Student t-test was performed to determine p-values (p-value > 0.05, ns).

Figure S4 :
Figure S4: Scheme of metastasis suppressor screening approach and validation of metastasis-related genes on chr8p in HCC68 cells.(A) Graphical scheme of identification of metastasis suppressor candidate genes on chr8p.(B) RNAi migration screen of chr8p candidate metastasis suppressors in HCC68 cells.Exemplary transwell migration images (top) are shown with respective quantification (bottom) of cell migration in four independent experiments.Knockdown was performed with two different siRNAs targeting each gene and quantified relative to Allstar and siGFP control (siRNA #1light grey, siRNA #2dark grey).Data are shown as floating bars with line indicating median and single dots representing each replicate of four independent experiments.(C) Western blot of HLF cell clones after transfection with empty vector (CTRL) or target gene overexpression (OE) and detection of HA tag and GAPDH or DLC1 and Vinculin.(D) Representative images of transwell migration assay in chr8pWT or chr8pLOH HCC68 cells after transfection with empty vector (CTRL) or target gene overexpression vectors (MSRA-HA, NAT1-HA, PPP2CB-HA, DLC1-V5).(E) Quantification of transwell migration in chr8pWT and chr8pLOH HCC68 cells after gene overexpression.Data are represented as mean ± SD of four independent experiments shown by single dots.(F) Heatmap of metastasis-associated gene expression after siRNA-mediated target gene knockdown in HCC68 cells compared to Allstar control (RT-qPCR data) and of chr8pWT and chr8pLOH HLF cells (RNAseq data).Z-scores are shown for gene expression relative to Allstar control (RT-qPCR) and relative to mean gene expression (RNAseq).Two-way ANOVA was performed for comparison of multiple groups.P-values are indicated above the graphs (pvalue > 0.05, ns).

Figure
Figure S5: Extended data of the CRISPR/Cas9 knockout screen and the chr8pLOH DepMap analysis.(A) Ranked gene essentiality scores of the CRISPR knockout screen in chr8pWT (left) and chr8pLOH (right) HLF cells.(B) Analysis flowcharts for the CRISPR knockout screen and DepMap analyses.(C) Scatter plot representation depicting gene essentiality scores in chr8pWT and chr8pLOH cells at day 7 (left) and mean gene essentiality scores of chr8pWT and chr8pLOH groups in the DepMap liver cancer (center) and pan-cancer (right) datasets.

Figure S6 :
Figure S6: Validation of NUDT17 dependency in chr8pLOH cells.(A) Growth curve for HCC68 chr8pWT and chr8pLOH cells after transduction with non-targeting sgRNA (NTsgRNA) or sgNUDT17 and cell viability measurement by resazurin assay for four consecutive days.Measurements of two independent sgRNAs for NUDT17 were combined.Out of four independent replicates, one representative growth curve is shown.Data are represented as mean ± SD of technical triplicates.(B) Relative cell viability of chr8pWT and chr8pLOH HCC68 cells following NUDT17 knockout 96 h post seeding.Data are represented as mean ± SD of four independent experiments with each dot representing the mean of one experiment.Colony formation of chr8pWT and chr8pLOH (C-D) HCC68 and (E-F) HLE cells.Cells were transduced with either NTsgRNA or two independent sgRNAs targeting NUDT17 and cultured for 14 days.Representative images of four replicates are shown.Quantification of colony area after NUDT17 knockout in chr8pWT and chr8pLOH (D) HCC68 and (F) HLE cells relative to NTsgRNA transduction.Data are represented as mean ± SD of four independent experiments with each dot representing the mean of one experiment.Two-way ANOVA was performed for comparison of multiple groups.P-values are indicated above the graphs (p-value > 0.05, ns).

Figure S7 :
Figure S7: Analysis of NUDT17 and NUDT18 deregulation.(A) NUDT17 and NUDT18 gene expression in four chr8pWT and four chr8pLOH cell clones as determined by RNAseq.(B) NUDT18 gene expression in TCGA-LIHC for normal liver tissue (NT) and HCC tumor (T) samples.(C) Kaplan-Meier survival curve of TCGA-LIHC patients with high (red, N 185) or low (blue, N = 184) NUDT18 gene expression.Hazard ration (HR) with 95% confidence interval and p-values were calculated by log-rank test.(D) Colony formation of chr8pWT and chr8pLOH HLF cells.Cells were transfected with siPools targeting NUDT17, NUDT18 or negative control (siCTRL) and cultured for 14 days.Representative images of five replicates are shown.Quantification of colony area after NUDT17 and NUDT18 knockdown in chr8pWT and chr8pLOH cells relative to siCTRL.Data are represented as mean ± SD of five independent experiments with each dot representing the mean of one experiment.(E) NUDT17 and NUDT18 gene expression in pTRIPZ-NUDT18-infected chr8pLOH HLF and HCC68 cells after transfection with siNUDT17 or siCTRL and treatment with doxycycline (DOX).(F) NUDT17 and NUDT18 gene expression in chr8pWT HLF and HCC68 cells after siPool mediated knockdown of NUDT17 and NUDT18 alone or in combination.Gene expression was determined by quantitative RT-PCR and analyzed with the comparative Ct method.Two-way ANOVA was performed for comparison of multiple groups.P-values are indicated above the graphs (p-value > 0.05, ns; p < 0.001 ***).